Correlation Of Degron-Dependent Proteolysis Of A Toxin In-Vivo With Enzyme Kinetics Of A Model Substrate In-Vitro
Date
2021-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
ATP-dependent proteases contribute to cellular viability by maintaining protein quality throughout the cell, and regulating cellular function through targeted protein degradation. These large enzymatic complexes hydrolyze ATP in order to power protein mechanochemical unfolding, translocation and degradation. The recognition of protein substrates is vital initial step in the process of proteolysis. Many proteases recognize substrates based on unstructured terminal sequences termed degrons. Developing an understanding of degron recognition is vital for the characterization of protease function within cells. One of the most widely validated degrons is the ssrA tag in the model organism Escherichia coli, which is recognized as a degradation signal by the ATP-dependent proteases ClpXP. Mutations in the last amino acid of this tag have been shown to significantly weaken the KM for proteolysis, while leaving the Vmax unaffected. I have cloned, expressed, and purified three GFPCP7-ssrA constructs with variation in the last amino acid of the ssrA tag: YALAA (wild-type), YALAS, and YALAD. Based on in-vivo experiments, it was hypothesized that in an in-vitro proteolysis assay with ClpXP, the YALAA construct would have the tightest KM, followed by YALAS, and lastly YALAD. The results confirmed our hypothesis, as the YALAA construct had a significantly tighter KM than the remaining constructs. The lack of comprehensive knowledge regarding degron-based recognition can be attributed to the inefficiency of utilizing in-vitro proteolysis assays for the purpose of degron discovery. However, these results can be used to affirm the correlation between an in-vivo toxin-based platform that assesses degron strength based upon cellular growth, and the in-vitro method using proteolysis assays to evaluate degrons.
Description
Keywords
In-vivo, In-vitro, Degrons, Proteolysis